Alzheimer's disease (AD) is a neurodegenerative disorder and the most common form of dementia in the elderly (reviewed in Hardy & Selkoe, (2002) Science 297(5580):353-6; Mattson, (2004) Nature 430(7000):631-9 and Walsh & Selkoe, (2004) Neuron 44(1):181-93). AD is characterized clinically by a progressive loss in cognitive function, including memory impairment, deterioration in language and visuospatial functions, and alterations in personality and behavior. Pathologically, AD is characterized by the presence of β-amyloid plaques and neurofibrillary tangles in the cortex and hippocampus. Amyloid β peptide (Aβ) is the main component of plaques and tau is the main component of tangles. Genetic evidence from familial early onset forms of AD (FAD) suggests that aggregation and accumulation of Aβ, specifically Aβ-42, initiates the cascade of events leading to neuropathology and dementia. Further support for the amyloid hypothesis is provided by transgenic mouse models where overproduction of Aβ1-42 recapitulates many of the hallmarks of AD including formation of plaques and cognitive deficits. Recent evidence from a triple transgenic mouse model of AD suggests that Aβ aggregation and accumulation proceeds and initiates tangle formation (Oddo et al., (2003) Neurobiol. Aging 24(8):1063-70; Oddo et al., (2004) Neuron 43(3):321-32; Oddo et al., (2003) Neuron 39(3):409-21).
Aβ is generated by proteolytic processing of APP by two enzymes, β-amyloid cleavage enzyme (BACE) and gamma secretase (γ-secretase). γ-secretase is a complex comprised of four proteins: presenilin (presenilin-1 or -2), nicastrin APH-1 and PEN-2 (De Strooper, (2003) Neuron 38(1):9-12). Presenilin-1 and -2 contain transmembrane aspartyl residues that have been shown to be essential for catalytic processing activity of the complex. The majority of the mutations linked to the early onset, familial form of AD (FAD) are associated with either PS-1 or PS-2. γ-secretase appears to have the capacity to process any type I transmembrane protein that has undergone ectodomain shedding (Struhl & Adachi, (2000) Mol. Cell. 6:625-636). In addition to APP, γ-secretase also been shown to cleave a number of other substrates including the Notch family of receptors (1-4), the Notch ligands Delta-1 and Jagged-2, E-Cadherin, ErbB4 and CD44 (De Strooper, (2003) Neuron 38(1):9-12). Genetic evidence indicates that the γ-secretase complex is critically required for Notch signaling and function, at least in the context of the developing embryo (Struhl & Greenwald, (1999) Nature (London) 398(6727): 522-525; Ye et al., (1999) Nature (London) 398(6727):525-529; Levitan & Greenwald, (1995) Nature (London) 377(6547): 351-5; Levitan & Greenwald, (1998) Development (Cambridge, U. K.) 125(18):3599-3606; Huppert et al., (2000) Nature 405:966-970; Donoviel et al., (1999) Genes Dev. 13(21):2801-2810; Herreman et al., (1999) Proc. Natl. Acad. Sci. U.S.A. 96(21):11872-11877). The physiological role of γ-secretase-mediated cleavage of Notch in the adult and of the other substrates is not known.
Notch is an evolutionarily conserved and widely expressed single-span type transmembrane receptor that plays a prominent role in regulating cell fate decisions in the developing embryo (reviewed in Artavanis-Tsakonas et al., (1999) Science 284(5415):770-6 and Kadesch, (2000) Exp. Cell Res. 260(1):1-8.). The role of Notch in the adult is less clear but Notch proteins are expressed in various adult tissues and are thought to play a role in regulating stem cell differentiation. Four Notch genes have been identified in mammals (Notch 1-4); all four Notch proteins are cleaved by γ-secretase (Mizutani et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98(16):9026-9031). Notch activation is induced by binding, in trans, to the Delta/Serrate/LAG2 family of transmembrane ligands. Notch signal transduction is mediated by three cleavage events: (a) cleavage at Site 1 in the extracellular domain (Logeat et al., (1998) Proc. Natl. Acad. Sci. U.S.A. 95(14):8108-12); (b) cleavage at Site 2 just N-terminal to the extracellular/transmembrane domain boundary following ligand binding (Brou et al., (2000) Mol. Cell. 5(2):207-216; Mumm et al., (2000) Mol. Cell. 5(2):197-206; Pan & Rubin, (1997) Cell 90(2):271-80); and (c) cleavage at Site 3 (S3) within the transmembrane near the transmembrane/cytoplasmic domain boundary (Schroeter et al., (1998) Nature (London) 393(6683):382-386; Kopan et al., (1996) Proc. Natl. Acad. Sci. U.S.A. 93(4):1683-8). Site 3 cleavage is required for release of the Notch intracellular domain (NICD) and is mediated by γ-secretase (Struhl & Greenwald, (1999) Nature (London), 398(6727):522-525; Levitan & Greenwald, (1998) Development (Cambridge, U. K.) 125(18):3599-3606; Mizutani et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98(16):9026-9031; Saxena et al., (2001) J. Biol. Chem. 276(43):40268-73; De Strooper et al., (1999) Nature (London) 398(6727):518-522). NICD activates transcription mediated by the CBF1/Su(H)/LAG-1 family of DNA-binding proteins and induces expression of various genes including HES-1 (Jarriault et al., (1998) Mol. Cell. Biol. 18(12):7423-31; Ohtsuka et al., (1999) EMBO J. 18(8):2196-207). NICD-regulated transcription is thought to be a key component of Notch-mediated signal transduction.
The development of γ-secretase inhibitors to block APP cleavage and Aβ generation is one therapeutic approach for the treatment of AD. This approach, however, is beset by the potential for mechanism-based toxicity due to inhibition of Notch processing. Indeed, Notch-related toxicities have been observed in studies where animals have been dosed subchronically with γ-secretase inhibitors (Wong et al., (2004) J. Biol. Chem. 279(13):12876-82; Searfoss et al., (2003) J. Biol. Chem. 278(46):46107-16; Milano et al., (2004) Toxicol. Sci. 82(1):341-58). One toxicity consistently observed following three or more days of treatment is an intestinal goblet cell metaplasia (Wong et al., (2004) J. Biol. Chem. 279(13):12876-82; Searfoss et al., (2003) J. Biol. Chem. 278(46):46107-16; Milano et al., (2004) Toxicol. Sci. 82(1):341-58). This lesion is similar to the phenotype observed in Hes-1 KO mice (Jensen et al., (2000) Nature Genet. 24(1):36-44), suggesting that the inhibitor-induced lesion is linked to inhibition of Notch signaling through Hes-1. Another molecule mediated by Notch is Trefoil factor-3 (TFF-3), also known as Intestinal Trefoil factor (ITF). TFF-3 is abundantly expressed by goblet cells in the duodenum demonstrates remarkable resistance to both proteolytic and thermal degradation. In addition to the GI lesion, alterations in lymphocyte development have also been noted after 5-15 days of dosing, including thymus atrophy, reductions in thymocyte numbers and alterations in thymocyte differentiation. These results are also consistent with inhibition of Notch processing and inhibition of it's role in regulating lymphocyte development (Wong et al., (2004) J. Biol. Chem. 279(13):12876-82).
Despite the potential for mechanism-based toxicity, γ-secretase inhibitors have been developed with some or complete specificity for inhibiting APP processing (Petit et al., (2003) J. Neurosci. Res. 74(3):370-7; Weggen et al., (2001) Nature 414(6860):212-6; Barten et al., (2005) J. Pharmacol. Exp. Ther. 312(2):635-43). In order to screen such inhibitors in vivo, it is desirable that biomarkers be developed that can be employed to monitor safety with respect to potential Notch-related toxicities.
A set of indicators that could be used to gauge toxic effects in vivo would therefore be of great value. A single set of reagents and standards could be used to evaluate test compounds from initial screening, through testing in pre-clinical (e.g., drug discovery) species, and potentially in clinical trials. Such universal indicators of toxicity preferably meet several criteria. First, they preferably are able to correctly identify toxic compounds with diverse mechanisms of action, including various chemical classes/chemotypes. Second, changes in these biomarkers are preferably consistent, quantifiable and reflect the degree of toxic insult. Third, assays are generally adaptable to high throughput technologies without becoming prohibitively expensive. Fourth, in vivo sample collection is preferably non- or minimally invasive, i.e. urine or blood is collected. Fifth, since there may be a need to analyze archival samples, it is preferable that the biomarker is stable.
Thus, what is needed is a method of determining in vivo the ability of a test compound known or suspected to modulate Notch processing mediated by γ-secretase. As such, this invention demonstrates that TFF-3 can be used as a such a marker.